Hepatocellular carcinoma (HCC) is an unfortunate and predictable consequence of hepatitis C virus (HCV) infections, defining it as a significant global human heath problem. There is no vaccine against HCV, and current treatments fail to cure HCV infections in nearly half of the cases. The consequence is that the untreated infected cells continue on their course toward cirrhosis and HCC. The goal of this project is to test the promise of a synergistic double hit against HCC by using RNA molecules to activate both innate immune and cell death response pathways in liver. This goal is based on recent evidence demonstrating that these pathways share common proteins and jointly assemble at the mitochondrion. Prior work from our laboratory has identified RNAs derived from the 3' untranslated region of hepatitis C genomic RNA or its antisense sequence that are potent activators of the RIG-I mediated innate immune signaling pathway. These RNAs have a 5' triphosphate, are about 100 nucleotides in length, and are described as polyU/UC or polyAG/A. Unlike the membrane-bound toll-like receptors, RIG-I is a cytoplasmic RNA binding protein and pattern recognition receptor that recognizes RNAs with a 5' triphosphate, initiating a signaling cascade that culminates in the expression if Type I interferons, establishing an antiviral environment. To date, our work has focused primarily on understanding how RIG-I binds to viral RNA and how the signaling pathway is activated. We discovered that RNAs containing modified ribose groups (2'-F deoxyuridine, for example) bind to RIG-I, but do not activate signaling, in contrast to their unmodified counterparts. Recent evidence from our group shows that the modified RNAs are unique tools that can be used to dissect the steps in the signal transduction pathway to understand mechanisms and potentially identify points of therapeutic intervention. In this proposal, we extend our work to focus on determining if the RIG-I agonist RNAs (polyU/UC and polyAG/A) activate both innate immune signaling and cell death pathways. Part of the approach focuses on the innovative use of a novel primary human liver culture cell system that maintains many physiologic markers at normal levels for up to two weeks, permitting experimentation in a physiologically relevant model. The significance of our approach is that RIG-I agonist RNAs may serve as adjuvants to boost an innate immune response to limit HCV infection, while also stimulating cell death pathways to kill cells that are progressing toward tumor formation. Indeed, recent studies have shown that RIG-I agonists activate cell death signaling, and that melanoma cells are more susceptible than normal cells to the killing effects. Although toll-like receptor ligands of the innate immune system are being used a immunostimulants to treat clinical disease, related approaches that activate the RIG-I- like helicase receptors can now be considered. This proposal has the potential to foster new ideas about how both HCV infection and the liver cancer that often follows can be treated or prevented.